System and Method for Eluting and Testing Substance From Exhaled Aerosol Sample

ABSTRACT

A method recovering one or more nonvolatile compounds from an exhaled aerosol collected on an electrostatic filter membrane ( 205 ) comprises eluting the nonvolatile compounds from the filter with a polar solvent and collecting the eluate. The method is useful for processing samples collected from a portable breath analysis device comprising a sampling filter membrane for analysis.

FIELD OF THE INVENTION

The present invention relates to methods for eluting nonvolatilecompounds from a solid matrix. In particular, the invention is a methodfor eluting and optionally analyzing nonvolatile compounds collected ona membrane from exhaled air.

BACKGROUND OF THE INVENTION

Exhaled breath is commonly used in alcohol testing and today'stechnology makes it possible to perform on-site breath testing withlegally defensible results using infrared spectroscopy.

Testing DNA and testing for drugs, whether abused or usedtherapeutically, generally requires the taking of blood or urine samplesfor testing. Hair, sweat, and oral fluid can also used for drug and DNAtesting. Blood sampling is invasive and requires medically trainedpersonnel, and the test subject is often transported to a hospital orother specialized facility for sampling. These procedures are time andeffort consuming. The collection of urine samples may also be consideredintruding on personal integrity. Other problematic issues related tosamples and specimens taken from a subject include risk of diseasetransmission or infection and the risk of the subject exchanging samplesor using a clean sample from another subject to avoid detection ofillicit drugs.

The need to provide a non-invasive, not-specimen based apparatus, systemand/or method for detecting the presence (i.e. qualitative) ordetermining the quantitative amount of a substance in a subject is atleast partially met by the invention disclosed in PCT Application No.PCT/EP2012054180, the contents of which are incorporated herein byreference for all purposes. PCT/EP2012054180 discloses an apparatus,system and method for sampling the exhaled breath of a subject for adrug substance and/or a biomarker. Aerosols from the lungs of thesubject are collected on a sampling membrane and then analyzed for thepresence and optionally the amount of one or more nonvolatile compounds.

In DE19718924, breath is in a specific exhalation device conveyedthrough a fiber fleece. Large aerosol particles are caught in the fiberfleece by condensation. The device of DE19718924 comprises severalvalves which increase the resistance to blow through the device. Thefiber fleece is removed from the housing, folded into the upper part ofa centrifugal tube. The fiber fleece can be contaminated by thisprocedure. A standard elution solution, such as ELISA buffer or saltsolutions, is added to the tube. A total amount of lung substances fromthe caught aerosol is determined from the eluate obtained. Thedetermined total amount of lung substances allows for diagnosis andtherapy of diseases, such as asthma. However, the disclosure ofDE19718924 is not very detailed. The total amount of lung substancesobtained as a result is not very specific and allows only coarsediagnostics and doubtful therapy based on such diagnostics. Samplingtimes in a range of 5-15 minutes are rather long and consistency ofresults over the range is not demonstrated. The method and device ofDE19718924 do not appear to be very efficient and have a limitedapplicational range. Despite DE19718924 being published in 1998, nomajor improvement has been disclosed during 15 years. Hence a long feltneed exists for improvement.

The present invention provides a method for eluting nonvolatilecompounds from a sampling membrane in such a way that the elutedcompounds are easily assayed. The present method improves the yield andconsistency of recovery of nonvolatile compounds from sampling membranesused to collect aerosols from exhaled breath.

SUMMARY OF THE INVENTION

Accordingly, embodiments of the present invention preferably seek tomitigate, alleviate or eliminate one or more deficiencies, disadvantagesor issues in the art, such as the above-identified, singly or in anycombination by providing a method, according to the appended patentclaims.

According to one aspect of the invention, a method for recovery isprovided for collecting nonvolatile compounds from a sample taken fromthe exhaled breath of a subject for further analysis. The method maycomprise removing a sampling membrane from a housing of a breathsampling device.

Alternatively, the sampling membrane may be in place in a housing of abreath sampling device. Nonvolatile compounds may be eluted by passing apolar solvent through an inlet and an outlet in an adapted housing. Byleaving the sampling membrane in place and eluting through the housinglowers the risk of contamination of the membrane. It also provides amore convenient and fast way of handling a collected breath sample.

A sampling membrane comprising collected from exhaled breath andaerosols containing nonvolatile compounds of interest is eluted bypassing a polar organic solvent through the membrane using gravity aloneor under pressure or centrifugal force.

In some examples of the invention, the sampling membrane is a filtermembrane, preferably an electrostatic filter membrane, and mostpreferably an electrostatic membrane comprising nonwoven acrylic andpolyethylene fibers.

The sampling membrane may be a filter membrane comprising at least onelayer of non-woven filtration media with a specific weight in the rangeof 23 gm3 to 500 gm3, preferably in the range of 150 up to 300 gm3, andeven more preferably in the range of 200 up to 280 gm3 and may compriseat least one further layer that is a spunbonded carrier with a scrimweight of 10 to 20 gm3.

The polar solvents suitable for use in the method are miscible in water,are capable of dissolving water soluble and lipid soluble components ofthe exhaled aerosol, and are capable of eluting the nonvolatilecompounds of interest. Polar solvents meeting these criteria includemethanol, acetonitrile, isopropanol, and acetone.

The nonvolatile compounds of interest may originate from blood by amechanism of producing a gas phase in the alveoli or from other parts ofthe airways and are transferred from the lungs carried by an aerosol.The nonvolatile compounds may be medical drugs, drugs of abuse,metabolites of drugs, markers indicative of drug use, or naturallyoccurring markers of disease states or metabolic conditions of subjects.Analysis of nonvolatile compounds from sample membranes may includemass-spectroscopy (MS) and Surface Enhanced Raman Spectroscopy (SERS)and other sensitive analytical methods known in the art.

In some examples of the invention, the aerosol on the sampling membranemay contain nonvolatile compounds of at least one drug substance in theexhaled breath. Nonlimiting examples of drug substances includeamphetamines, ecstasy, Cannabis, THC, cannabinoids, opiates, heroin,morphine, 6-AM, cocaine, benzodiazepines, Propoxyphene, Methadone,Buprenorphine, Tramadol, LSD, Designer/Internet drugs, Kathinon, GHB,Meprobamat, Z-drugs, tryptamines, and anabolic steroids.

MS and SERS are the preferred analyzing methods with liquidchromatography /MS (LC/MS) being preferred over gas chromatography /MS(GC/MS).

Further examples of the invention are defined in the dependent claims,wherein features for the second and subsequent aspects of the inventionare as for the first aspect mutatis mutandis.

Some known nonvolatile biomarker that may be transported by aerosols inexhaled breath is comprised in the list comprising lipids, peptides,nucleotides, prostanoids, proteins, DNA or RNA. These biomarkers may beused for diagnosis of diseases or illnesses, such as cancer (such aslung cancer), asthma, inflammation, infection (such as tuberculosis)and/or oxidative stress or other medical conditions.

The term “comprises/comprising” when used in this specification is takento specify the presence of stated features, integers, steps orcomponents but does not preclude the presence or addition of one or moreother features, integers, steps, components or groups thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other aspects, features and advantages of which examples ofthe invention are capable of will be apparent and elucidated from thefollowing description of examples of the present invention, referencebeing made to the accompanying drawings, in which

FIG. 1 is a schematic illustration illustrating an example of a portabledevice configured to collect a sample from exhaled breath of a subject;

FIG. 2 is a flow-chart illustrating a method for using a portable deviceconfigured to collect a sample from exhaled breath of a subject;

FIG. 3 is a flow chart of method steps for a recovery method accordingto the invention;

FIG. 4 illustrates a system for detecting the presence or determiningthe quantitative amount of at least one drug compound in exhaled breath;and

FIG. 5 illustrates typical chromatograms for four different drugs.

DESCRIPTION OF EMBODIMENTS

Specific embodiments of the invention will now be described withreference to the accompanying drawings. This invention may, however, beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will be thorough and complete, and willfully convey the scope of the invention to those skilled in the art. Theterminology used in the detailed description of the embodimentsillustrated in the accompanying drawings is not intended to be limitingof the invention. In the drawings, like numbers refer to like elements.

In FIG. 1 is a schematic illustration showing an example of a portablesystem configured to collect a sample from exhaled breath of a subject.Sampling system 20 is configured to collect a sample from exhaled breathof a subject 201. The subject exhales through a mouthpiece and a tubularelement 202 being in flow communication with a housing 204 via at leastone inlet 203. The mouthpiece and tubular element 202 may be of samesize or type as conventionally used for alcohol-testing. Preferably, themouthpiece and tubular element 202 are configured to mainly permitaerosols to pass through.

The housing 204 can be made of any suitably material or combinationsthereof such as, metal, plastic, glass or ceramics and comprises asampling membrane 205, being a fibre membrane, for collecting aerosolscomprising non-volatile drug substances from the exhaled breath. Theexhaled breath exits the housing through at least one outlet 206. Theportable system may be sealed after being used and sent to a laboratory207 to be analyzed. Analysis of the amount of illicit drugs may beperformed using any suitable method with Surface Enhanced RamanSpectroscopy (SERS) and mass-spectroscopy (MS), for example Liquidchromatography MS (LCMS), being most preferred.

The sampling membrane can be placed anywhere in the housing 204.Preferably the sampling membrane 205 is fastened to the inner walls ofthe housing 204 and stretched over the cross-section of the housing. Thehousing 204 is preferably constructed out of two or more parts, a firstpart with an inlet 203 and one part with at least one outlet 206.

The mouthpiece and tubular element 202 are detachable and in fluidcommunication with the housing 204 via the inlet 203. The tubularelement 202 functions as a pre-filter for filtering out saliva and/ormucus and/or large particles and/or aggregates from the exhaled breath.Thus a cleaner sampling membrane, such as a filter membrane, to beanalyzed is obtained, which may result in better and more accurateanalysis. Another advantage of using a tubular element 202 is to preventcontamination between subjects and samples.

The exhaled breath will, after flowing through the tubular element 202,travel into the housing 204 and be brought into contact with thesampling membrane 205, preferably a filter membrane.

The drug substances, being non-volatile compounds conveyed by aerosolsin the exhaled breath, are collected by the sampling membrane 205 as theexhaled breath is permeated through the sampling membrane 205.

The sampling membrane 205, being a filter membrane is not to be confusedwith an electronic or chemical sampling unit and/or trap. The samplingelement 205 is a physical entity on which the drug substance iscollected. Collection may, in different embodiments, be based on variousprinciples, singly or in combination, comprising depositing, catching,fastening, and condensing of non-volatile constituents on the samplingelement 205.

Using a filter membrane allows for a low pressure drop through theportable sampling system, making it easy and comfortable to exhalethrough the device. This is important for subjects experiencing problemswith their lungs.

In an example of a portable sampling system, the filter membrane isplaced to block the path for the exhaled breath through the portablesystem.

There are many possibilities for fastening the filter membrane, forexample, using a separate support structure to retain the filter, whichmay be an element that is either fastened to the inside walls of thehousing 204 or a segment being slid onto a first part of the housingduring the assembly before the second part of the housing is mounted.

Alternatively and/or additionally, the filter membrane may be fasteneddirect onto the inside walls of the housing 204, either by glue or byheat and thereby melting a small part of the edge of the filtermembrane.

The second part of the housing is either screwed or slid onto the firstpart of the housing. The second part comprises at least one outlet 206.Alternatively and/or additionally, the housing 204 may comprise at leasttwo outlets 206. This will aid to avoid a subject being tested to blockthe outlet and thereby blowing up the volume measuring element withminimum exhaled breath being permeated through the filter membrane

The sampling element 205 is preferably a filter membrane made ofsynthetic and/or half-synthetic fibers for the exhaled breath to diffusethrough. The filter membrane has a structure that catches the aerosolsand thereby collects the drug substances being exhaled while letting gaspass through. The aerosol particles comprise the non-volatile drugcompounds. Preferably, the filter membrane is operable to collect theaerosols from the air with a high volumetric capacity while maintaininga low pressure drop across the filter substrate. Alternatively and/oradditionally, in some examples the filter membrane may be anelectrostatic filter made of fibers. Alternatively and/or additionally,the filter membrane may be of a non-woven polymeric fibrous that may bean electret.

Alternatively and/or additionally, the filter membrane may be anelectrostatic filter being preferably a non-woven filter membranecomprising a blend of acrylic fibers and polypropylene fibers. Thefilter membrane may have a density (sometimes called grade or weight) inthe range 23 up to 500 gm2. But it has been shown that the range 130 upto 300 gm2 is preferred. Even more preferably is the range 200 up to 280gm2.

Alternatively and/or additionally, the non-woven layer may be attachedto at least one further layer. Further layers can be used to enhance afilter membrane's physical properties or improve filtration performance.The extra layer can be a carrier, preferably a polypropylene spunbondedcarrier. The spunbond carrier may have a scrim weight in the range 10 upto 20 gm2, preferably 15 gm2.

For example a three layered filter membrane comprising one non-wovenlayer with a density of 250 gm2 and two layers being carriers each witha scrim weight of 15 gm2 will have an air flow resistance of about 36 Paat 9.5 m/min media velocity. Alternatively and/or additionally, thefilter membrane may be corrugated to enhance the filtering area within agiven housing volume.

The portable system combined with the method of eluting the content ofthe filter membrane is configured and adapted to have a sensitivity forillicit drugs high enough to obtain results of a standard to be used asproof in a court of law.

The collected aerosols may comprise nonvolatile compounds of at leastone drug substance in the exhaled breath. The drug substance may, by wayof example, be an amphetamine, ecstasy, Cannabis, THC, a cannabinoid, anopiate, heroin, morphine, 6-AM, cocaine, a benzodiazepine, Propoxyphene,Methadone, Buprenorphine, Tramadol, LSD, a designer/Internet drug,Kathinon, GHB, Meprobamat, a Z-drug, a tryptamine, or an anabolicsteroid. Other illicit drugs (drugs of abuse) and therapeutic drugs notincluded in the list may also be detectable due to similar interchangeswith the human body as the above mentioned illicit drug substances.

Alternatively and/or additionally, non-volatile compounds may includebiomarkers transported by aerosols in exhaled breath. These biomarkersmay comprise lipids, peptides, nucleotides, prostanoids, proteins, DNAor RNA and may be used for diagnosis of diseases or illnesses, such ascancer (such as lung cancer), asthma, inflammation, infection (such astuberculosis) and/or oxidative stress or other medical conditions. Thesebiomarkers may be collected and recovered using the system and methodsdescribed herein.

FIG. 2 is a flow-chart illustrating a method 50 for using a portablesystem configured for collecting a sample 502 from exhaled breath from asubject. Briefly, FIG. 2 illustrates detecting the presence and/ordetermining the quantitative amount 503 of at least one illicit drugsubstance or compound in a collected sample. The illicit drug compoundsare collected as aerosols, being non-volatile particles and/orcompounds, from the exhaled breath. The method comprises: a subjectexhaling 501 into the invented portable system; a sampling unit collectsa sample 502 comprising drug substances or compounds; the collectedsample is extracted from the sampling membrane and analyzed 503 using MSor SERS.

With reference to FIG. 2, a subject exhales 501 in and out; preferablythe subject exhales into the portable system until a specific volume ofexhaled breath has been passed through the sampling membrane, preferablya filter membrane.

Alternatively and/or additionally, the subject may exhales either for acertain time or for a fixed number of times, such as 1 to 10 times, intoa portable system. When breathing a fixed number of times, eachexhalation may be set to last for a fixed time.

To measure a specific volume, one preferred method is to use a portlocated on the mouthpiece or between the mouthpiece and the inlet of thehousing. A portion of the exhaled breath is extracted 504 through theport and inflates an element, such as a non-elastic bag. Hence when thebag is full, the volume of said bag will be proportional to the volumepassed through the sampling membrane.

Alternatively and/or additionally, a bag with elastic properties may beused.

A deep breath is preferred to obtain exhaled breath from deep lying lungportions, such as the central or peripheral lung regions.

The exhaled breath is then collected 502 by the sampling element, i.e.an easy to breathe through filter membrane, suitable for collecting drugsubstances before it exits the system. The filter membrane is preferablymade of synthetics and/or half synthetic fibers. Preferably, the filtermembrane has electrostatic-properties. Using a filter membrane creates asmall, light-weight and easy to use method that can be used everywhereby anyone to detect if a subject is under influence of an illicit drug.The sampling method is of such high quality that the obtained resultsare of a court approved standard. These results are obtained using amethod that is neither invasive, e.g. Blood sampling, nor intruding onpersonal integrity, e.g. urine sampling. Hence the known drawbacks withthese methods are prevented.

After being used, the housing of the sampling system is sealed off bysealing the inlet and the outlet and the device may be sent to alaboratory, where the collected compounds in the filter are recovered ananalyzed.

FIG. 3 illustrates a method 30 for recovering one or more nonvolatilecompounds from a sampling filter membrane. The fibers are preferablynonwoven and filter is most preferably a felt or a matted material. Thefilter is preferably an electrostatic filter and most preferablycomprises acrylic fibers and polypropylene fibers. The method comprisescontacting 301 the filter with an amount of a polar solvent capable ofneutralizing the electrostatic charge on the filter. The contacting ofthe filter with the polar solvent elutes the nonvolatile compounds fromthe sampling filter to form an eluate. Preferred polar solvents includemethanol, acetonitrile, isopropanol, acetone, and combinations thereof,with methanol and acetonitrile being most preferred for their relativeease of handling. The polar solvents are miscible in water, are capableof dissolving water soluble and lipid soluble components of the aerosol,and thereby eluting the nonvolatile compounds of interest from thefilter membrane. Polar solvents meeting these criteria include methanol,acetonitrile, isopropanol, and acetone. Methanol and acetonitrile arepreferred polar solvents in combination with filter membranes comprisingacrylic and polyethylene fibers. Preferably, the polar solvents are usedin pure form, i.e. in no mixture with other compounds or solvents. Puresolvents are e.g. pure methanol, or pure acetonitrile.

The amount of solvent used depends on the size of the filter and shouldbe enough to elute the nonvolatile compounds but should notunnecessarily dilute the compounds. By way of example, the amount ofsolvent may be between 0.02 ml and 20 ml. Contacting may be accomplishedby washing the filter with the solvent, for example by passing an amountof the solvent through the filter. The filter 205 is preferably in thehousing 204 of the sampling device with the solvent being passed thoughthe device from one of the inlet 203 or outlet 206 to the other of theinlet 203 or outlet 206 of the device. Alternatively, the filter 205 maybe removed from the device and placed in a column for elution with thesolvent by gravity or with pressure. The filter may alternatively besoaked in the solvent before being placed in a centrifuge tube orcolumn.

After the solvent is contacted with the filter membrane, the eluate iscollected 302 in a suitable container. Collection may be accomplished,for example, by gravity flow from a filter in a device or,alternatively, saturating the filter with solvent and placing the devicein a centrifuge with a collecting vessel positioned to receive theeluate during centrifugation. The eluate may also be collected byapplying solvent to a sampling device and applying pressure to force thesolvent through the filter and into a collecting vessel. Collection mayalso be accomplished by gravity, pressure, or centrifugation from afilter that has been removed from a sampling device and placed in acolumn or tube for elution by gravity, pressure, or centrifugation.

The collected eluate may be stored or optionally delivered 303 to anassay system capable of detecting (qualitative analysis) and/ormeasuring (quantitative analysis) one or more nonvolatile compounds inthe eluate. Any suitable assay system may be used, with LC/MS, GC/MS,and SERS being most preferred. MS may include, for example,time-of-flight MS, quadrupole ion trap MS, tandem MS, electrosprayionization MS, and matrix-assisted laser desorption/ionization MS.Depending on the assay system being used, the eluate may be pretreatedor concentrated before delivery to the assay system. For LC/MS, theeluate may be loaded into the liquid chromatography column, optionallyafter reducing the volume of the eluate by evaporation of the solventwith or without partial vacuum. For SERS, the eluate may be placed on aSERS substrate comprising, for example, gold or silver colloids,optionally after reducing the volume of the eluate. The eluate mayalternatively be evaporated to dryness to form a dry residue that isresuspended in the same or a different solvent or combination ofsolvents before application to an assay system.

FIG. 4 illustrates a system 60 for detecting the presence or determiningthe quantitative amount of at least one drug compound in exhaled breath.The system comprising a portable drug testing device 602 for handheldcollection of a sample from exhaled breathe from a subject 601. Thedevice 602 could be according to any embodiment described herein.Further, the system 60 comprises a sensor unit 603 for analyzing thecollected compounds in the testing device 602. The sensor could be anyknow type of sensor but preferably mass spectroscopy or SERS. To be ableto analyze the collected sample the content of the filter membrane inthe device 602 is extracted. This could be done for example by using asolvent to be analyzed using the sensor unit 603.

An advantage is that the test may be performed almost anywhere at a lowcost and short lead time before obtaining a result.

EXAMPLE 1

Table 1 shows the results of recovering a variety of nonvolatilecompounds from an electrostatic filter membrane comprising acrylicfibers and polyethylene fibers by elution with methanol. The eluateswere analyzed using LC-MS/MS with reference solution. Recovery and assayfor each nonvolatile compound analyte was performed in triplicate.Typical chromatograms for some drugs when analyzing the eluates usingLC-MS/MS equipment are illustrated in FIG. 5. From the top to the bottomthe chromatogram shows peaks for, amphetamine 70, 75; Morphine 80, 85;Diazepam, 90, 95; and THC, 100, 105. As there is a linear relationbetween concentration (pg/filter) and the area ratio of analyte tointernal standard for all compounds the amount of drug in the filter maybe determined based on calibration curves from reference solutions.

TABLE 1 Recovery of nonvolatile compounds by methanol elution AnalyteRecovery (%) Analyte Recovery (%) Amphetamine 101 Methamphetamine 107THC 95 Morphine 98 Codeine 97 MDMA 93 Benzoylecgonine 95 Cocaine 95Buprenorphine 116 Methadone 115 Oxazepam 106 Flunitrazepam 1076-Acetylmorphine 97 Diazepam 105

The present invention has been described above with reference tospecific embodiments. However, other alternatively and/or additionallyembodiments than the above described are equally possible within thescope of the invention. Different method steps than those describedabove, performing the method by hardware or software, may be providedwithin the scope of the invention. The different features and steps ofthe invention may be combined in other combinations than thosedescribed. The scope of the invention is only limited by the appendedpatent claims.

1. A method for recovery of non-volatile compounds from an electrostaticfilter membrane, comprising: providing said electrostatic filtermembrane containing an exhaled aerosol comprising a nonvolatilecompounds; washing said electrostatic filter membrane with a polarorganic solvent to form an eluate; and collecting said eluate comprisingsaid nonvolatile compounds.
 2. The method according to claim 1, whereinsaid electrostatic filter membrane is fitted in a housing comprising atleast one inlet and at least one outlet, wherein said housing is part ofa sampling system for exhaled breath.
 3. The method according to claim2, further comprising pouring said solvent in one of said inlet oroutlet and collecting said eluate through the other of said inlet oroutlet.
 4. The method according to claim 1, wherein said filter membranecomprises at least one layer of non-woven filtration media with aspecific weight in the range of 23 g/m3 to 500 g/m3.
 5. The methodaccording to claim 4, wherein said electrostatic filter membranecomprises at least one further layer, said further layer being aspunbonded carrier with a scrim weight of 10 to 20 g/m3.
 6. The methodaccording to claim 4, wherein said electrostatic filter membrane has afilter surface to be passed by exhaled gas of approximately 800 mm2 anda pressure drop of 36 Pa at 9.5 m/min media velocity.
 7. The methodaccording to claim 1, wherein said electrostatic filter membranecomprises a blend of acrylic fibers and polypropylene fibers.
 8. Themethod according to claim 1, wherein said aerosols comprise nonvolatilecompounds of at least one drug substance in said exhaled breath; andwherein said drug substance is selected from the list comprisingAmphetamine, ecstasy, Cannabis, THC and cannabinoids, Opiates, heroin,morphine, 6-AM, Cocaine, Benzodiazepines, Propoxyphene, Methadone,Buprenorphine, Tramadol, LSD, Designer/Internet drugs, Kathinon, GHB,Meprobamat, Z-drugs, Tryptamines, Anabolic steroids, or combinationsthereof.
 9. The method according to claim 1, wherein said aerosolscomprise nonvolatile compounds of at least one biomarker in said exhaledbreath and wherein said biomarker is selected from the list comprisinglipids, peptides, nucleotides, prostanoids, proteins, DNA, RNA, orcombinations thereof.
 10. The method according to claim 1, furthercomprising contacting said collected eluate with a SERS surface.
 11. Themethod according to claim 1, and further comprising evaporating saidsolvent from said eluate.
 12. The method according to claim 1, furthercomprising performing a qualitative and/or quantitative analysis of acontent comprising said non-volatile compounds on the SERS-surface usingSERS-technique.
 13. The method according to claim 1, further comprisinganalyzing said eluate using a mass spectroscopy method.
 14. The methodaccording to claim 1, wherein said solvent is methanol.
 15. The methodaccording to claim 1, wherein said solvent is acetonitrile. 16.(canceled)
 17. The method of claim 1, further comprising evaporatingsaid eluate at least partially.
 18. The method of claim 17, furthercomprising evaporating said eluate to dryness to form a dry residue tobe resuspended in a solvent before being analyzed.
 19. A method forrecovery of non-volatile compounds from an electrostatic filtermembrane, comprising: providing said electrostatic filter membranecontaining an exhaled aerosol comprising a nonvolatile compound; washingsaid electrostatic filter membrane with a polar organic solvent to forman eluate; collecting said eluate comprising said nonvolatile compound;and analyzing said eluate using an analyzing method.
 20. The method ofclaim 19, wherein the analyzing method is qualitative or quantitative.21. A method for recovery of non-volatile compounds from anelectrostatic filter membrane, comprising: providing said electrostaticfilter membrane containing an exhaled aerosol comprising a nonvolatilecompound; washing said electrostatic filter membrane with a polarorganic solvent to form an eluate; and collecting said eluate comprisingsaid nonvolatile compound; and evaporating said eluate at leastpartially.